A New Precolumn Derivatization LC‐MS/MS Method for the Determination of Methoxamine Enantiomers in Rat Plasma: Application to Stereoselective Pharmacokinetic Analysis

Methoxamine is a selective α-adrenergic receptor agonist used to manage hypotension during anesthesia and prevent cardiac arrhythmias. Erythro-methoxamine, a racemic mixture, is clinically administered in this form. Previous studies revealed that the (1R,2S)-enantiomer exhibits stronger pharmacological activity than the (1S,2R)-enantiomer, highlighting the need for stereoselective pharmacokinetic evaluation. Given the high sensitivity, excellent stereoselectivity, and resistance to racemization exhibited by (S)-N-(4-nitrophenoxycarbonyl) phenylalanine 2-methoxyethyl ester [(S)-NIFE], this study pioneered its novel application as the chiral derivatization reagent for methoxamine prior to chromatographic analysis. Based on precolumn derivatization, a sensitive, efficient, and straightforward LC-MS/MS method was developed to determine the two enantiomeric derivatives of erythro-methoxamine. DHBA was selected as the internal standard. After protein precipitation, the derivatives were analyzed in positive ESI-MRM mode. The method showed good linearity over 0.5-250.0 ng·mL^-1 with an LLOQ of 0.5 ng·mL^-1. Intraday and interday RSDs were below 9.7%, and recoveries exceeded 86.2%. The validated method was successfully applied to the first stereoselective pharmacokinetic study of erythro-methoxamine in rat plasma. Significant differences in AUC, t_1/2, Vd, and CL were observed, confirming its suitability for in vivo pharmacokinetic analysis and providing evidence for the stereoselective disposition of methoxamine enantiomers.